Capture of target substances such as proteins and other biomolecules on solid phases has been used to facilitate their subsequent identification, to study their manner of interacting with other substances, etc. Typical interactions are those between proteins, such as between antigens and antibodies, hormones and receptors, biotinylated molecules and biotin binding proteins, and the like. One method of capture is generally designated as blotting, where the target substance is applied, directly or by transfer from another medium, to a membrane such as nitrocellulose, polyvinyllidene difluoride (PVDF), or nylon.
Blotting is most frequently used in combination with known gel electrophoresis procedures. The target substance, e.g., an antigen, is first separated on a gel from other substances, typically by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The target substance is then transferred electrophoretically to a membrane. Subsequently, the target substance is associated, either directly or indirectly through an intermediate primary ligand, by an affinity interaction to a reporter probe, such as an antibody, having a label attached to it that is capable of providing a detectable signal. Radioactive, calorimetric, fluorescent, or enzymatic labels are commonly employed to provide the detectable signals which, in turn, indicate the presence and/or location of the target substance. Responses from radioactive and fluorescent labels are generally determined directly, while the response (luminescent, fluorescent, or calorimetric) elicited with enzymatic labels is indirect in that the detectable signal results from the action of the enzyme on an appropriate substrate.
Procedures for electrophoretic separation with subsequent blotting onto a membrane are referred to in the literature as Western blotting, Northern blotting or Southern blotting. Western blotting refers to the identification of antigens as target substances, while the latter two procedures refer to identification of target RNA and DNA sequences, respectively. More recently, a variation of Western blotting, referred to as Far Western blotting, has been used to characterize protein-to-protein interactions other than antigen to antibody interactions.
A drawback associated with Western type and other blotting techniques is that they require time consuming and cumbersome steps. These include transferring the target substance from the gel to the membrane on which the substance is immobilized and then blocking the membrane. Some of these steps, particularly the transfer and immobilization operations, may be detrimental to the protein being assayed. For example, a change in antigenic nature of a protein may prevent the corresponding antibody from binding and, therefore, detecting the target molecule. In addition, the pattern obtained on a membrane when a crude lysate is transferred may not be a true representation, since smaller molecular weight proteins transfer more efficiently than larger molecular weight proteins. Additionally, some proteins simply do not transfer well and, therefore, are not represented on the membrane at all.
Thus, in-gel procedures, in which detection is accomplished without removing the target substance from the gel, are desirable. While in-gel techniques for detecting target substances in polyacrylamide gels were reported even before the advent of the above described blotting techniques (Burridge, K. (1976) Proc. Natl. Acad. Sci., USA, 73, 4457-4461; Rosta, J. A.; Kelly, P. T.; and Cotman, C. W. (1977) Anal. Biochem., 80, 336-376; and Olden, K. and Yamada K. M. (1977) Anal. Biochem, 78, 483-490), the procedures were very time consuming, entailing lengthy fixation, incubation and wash steps, which generally took on the order of several days. Furthermore, in-gel techniques have been considered not sufficiently sensitive for the detecting target substances using large reporter probes, e.g., antigen-antibody interactions; the problem was insufficient penetration of the probe or primary ligand into the three-dimensional gel to achieve the required association of the target substance and probe to permit detection at low concentrations of target substance.
It would be desirable to provide an efficient method that is sensitive and useful for in-gel detection of biomolecules.